JP5214941B2 - Single probe molecular device and method for producing single probe molecular device - Google Patents

Description

  The present invention relates to a single probe molecular element for detecting a biomolecule, a method for manufacturing the same, and a method for detecting a biomolecule.

  In recent years, various biomolecule detection elements have been researched and developed as methods for specifically and comprehensively analyzing biomolecules such as nucleic acids and proteins. An important example of such a biomolecule detection element is a probe molecule-fixed fine particle in which a probe molecule for detecting a biomolecule is fixed on the surface of a nanometer to micrometer-size fine particle. So far, various biomolecule detection methods using probe molecule fixed fine particles have been reported. Focusing on the probe molecule-fixed metal fine particles in which the probe molecules are fixed on the surface of the metal fine particles among the probe molecule-fixed fine particles, for example, Patent Document 1 discloses that a probe molecule-fixed gold nano-particle is compared with an analysis target molecule fixed on a carrier substrate. A method for identifying and quantitatively evaluating molecules to be analyzed by binding particles and detecting the gold nanoparticles with an electron microscope or the like is disclosed. Patent Document 2 discloses a method for analyzing biomolecules utilizing the fact that gold nanoparticles having oligonucleotides immobilized on their surfaces exhibit different colors depending on their aggregation state.

  In order to quantitatively analyze biomolecules using probe molecule-fixed metal fine particles, it is important that the number of probe molecules immobilized on the surface of the metal fine particles is accurately known. Furthermore, in order to realize quantitative analysis of biomolecules at the single molecule level, it is indispensable to use metal fine particles with a single probe molecule fixed on the surface. The probe molecule-fixed metal fine particles used in conventional known examples are produced by mixing and reacting metal fine particles and probe molecules having functional groups that strongly interact with the metal fine particles at a predetermined mixing ratio. Therefore, the probe molecule-fixed metal fine particles produced by this method are a mixture of metal fine particles having a distribution in the number of probe molecules immobilized on the surface. In order to obtain single probe molecule-fixed metal fine particles, it is necessary to separate and purify from a mixture of the metal fine particles.

  Non-patent document 1 discloses a method utilizing gel electrophoresis as a method for obtaining single probe molecule-fixed metal fine particles. In this method, gold nanoparticles and probe DNA having a thiol group at the 5 'end are mixed and reacted at a predetermined molar ratio to immobilize the probe DNA on the surface of the gold nanoparticles. At this time, a mixture of gold nanoparticles in which the number of probe DNA immobilized on the surface is 0, 1, 2, 3, and 3 or more is obtained. When gel electrophoresis is performed on this mixture of gold nanoparticles, the migration distance in the gel increases in order from the gold nanoparticles with the smallest number of immobilized DNA. Therefore, gold nanoparticles can be separated according to the number of immobilized DNA, and single probe DNA-immobilized gold nanoparticles can be separated and recovered. However, separation and recovery of single-probe DNA-immobilized gold nanoparticles in this method is very complicated, and the recovery rate of single-probe DNA-immobilized gold nanoparticles is very low.

  Non-Patent Document 2 discloses a method for immobilizing probe DNA on the surface of metal fine particles via polymer molecules. In this method, a dextran molecule having a plurality of disulfide groups as functional groups is synthesized, and probe DNA is bound to the dextran molecule. A dextran molecule to which the probe DNA is bound is immobilized on the surface of the gold nanoparticle using a disulfide group. In this method, it is possible to fix the probe DNA to the surface of the gold nanoparticle via the dextran molecule, but there is a problem that the number of probe DNAs bound to one dextran molecule is distributed. Therefore, there is a need for a novel method that can easily and highly yield metal fine particles having a single probe molecule immobilized on the surface.

  On the other hand, among techniques for detecting biomolecules at a single molecule level, development of gene sequence analysis techniques by sequencing at a single DNA molecule level as disclosed in Non-Patent Document 3 has been actively promoted. In this method, a huge number of polynucleotides as specimens are immobilized on the surface of a carrier substrate, and a single-base extension reaction with a polymerase is performed using this as a template to form a complementary strand of the polynucleotide. Four types of nucleotides necessary for the single-base extension reaction process are labeled with different fluorescent dyes, and fluorescence detection is performed each time one base is extended to identify the introduced nucleotide. By repeatedly performing this fluorescence detection on each polynucleotide immobilized on the carrier substrate, the base sequence of the polynucleotide can be comprehensively decoded. This method requires single-molecule fluorescence detection during a single base extension reaction, and in order to obtain highly accurate results, a method of immobilizing a polynucleotide to be analyzed on a carrier substrate one molecule at a time, and There is a need for a method for increasing the sensitivity of fluorescence detection. The polynucleotide that is the actual specimen in DNA sequencing is messenger RNA (mRNA), which is a transcript of genomic DNA.

  As an example of a method for improving the detection sensitivity of fluorescence, Non-Patent Document 4 reports a method using the fluorescence enhancement effect by metal fine particles. In this report, silver nanoparticles are regularly arranged on a carrier substrate, and probe DNA is bound to this surface. When this probe DNA is reacted with a fluorescently labeled specimen and the carrier substrate is irradiated with excitation light, the free electrons of the silver nanoparticles cause resonance vibration (localized plasmon resonance), and the fluorescence is enhanced. This fluorescence enhancement effect enables highly sensitive detection of the specimen. Although this report shows that fluorescence enhancement occurs, it is not aimed at single-molecule fluorescence detection, so a large number of probe DNAs are immobilized on the surface of silver nanoparticles. Therefore, in order to realize highly accurate single molecule fluorescence detection using this method, it is necessary to use metal fine particles having a single probe molecule fixed on the surface.

JP 2006-153826 A JP 2006-288406 A Nano Letters Vol.1, No.1, p32 (2001) Chem. Commun., P1156 (2004) Proc.Natl.Acad.Sci.USA, Vol.100 (7), p3960,2003 Biochem. Biophys. Res. Comm. 306 p213 (2003)

  An object of the present invention is to detect a biomolecule at a single molecule level with high sensitivity and high accuracy by a fluorescence method. The problem to achieve this object is to fix the biomolecules to be analyzed one by one on the biomolecule detection element and to increase the fluorescence detection sensitivity of the biomolecule detection element.

  In the present invention, firstly, in order to capture the biomolecules to be analyzed one by one, metal fine particles in which one probe molecule is fixed on the surface by chemical bonding, that is, single probe molecule-fixed metal fine particles are manufactured. By using probe molecules having a plurality of functional groups that can chemically bond to the surface of the metal fine particles, single probe molecule-fixed metal fine particles are produced in a high yield. Next, single probe molecule-fixed metal fine particles are fixed on a carrier substrate to manufacture a single probe molecule element. This single probe molecule element can capture biomolecules to be analyzed one by one on the surface of the carrier substrate via single probe molecules of single probe molecule-fixed metal fine particles. In addition, it is possible to detect fluorescence of a single molecule with high sensitivity and high accuracy by using the plasmon resonance effect of metal fine particles by excitation light when detecting fluorescence near the probe molecule. .

  According to the present invention, it is possible to provide a single probe molecular element capable of detecting a biomolecule at a single molecule level with high sensitivity and high accuracy by a fluorescence method. In the single probe molecule element, since one molecule of probe molecule is immobilized on the surface of the metal fine particle immobilized on the surface of the carrier substrate, one molecule of the biomolecule to be analyzed is placed on the surface of the carrier substrate using the probe molecule. It is possible to capture one by one. In the fluorescence detection, the fluorescence is enhanced based on the resonance vibration of the excitation light and free electrons in the metal fine particles, and the fluorescence from a single fluorescent molecule can be detected with high sensitivity and high accuracy.

Embodiments of the present invention will be described below with reference to the drawings.
FIG. 1 shows an example of single probe molecule-fixed metal fine particles according to the present invention. A single probe molecule 101 for capturing only one biomolecule to be analyzed is fixed to the surface of the metal fine particle 102 by a plurality of chemical bonds. In FIG. 1, a single probe molecule 101 is fixed on the surface of a metal fine particle 102 by three chemical bonds. In addition, an adsorption inhibiting molecule 103 is immobilized by chemical bonding on the surface of the metal fine particle 102 to a portion where the single probe molecule 101 is not immobilized, in order to prevent nonspecific adsorption of various biomolecules. ing. In FIG. 1, spherical fine particles are shown as the metal fine particles, but the shape of the metal fine particles 102 may be a cylinder, a cone, a prism, a pyramid, or the like.

  FIG. 2 shows an example of a single probe molecular device according to the present invention. Single probe molecule-fixed metal fine particles 202 are fixed to the surface of the carrier substrate 201 by chemical bonding.

An example of a process for producing single probe molecule-fixed metal fine particles according to the present invention will be described with reference to FIGS. The manufacturing process of the single probe molecule-fixed metal fine particle includes the following six steps. Here, we will explain using DNA as a probe molecule. However, this manufacturing process not only provides nucleic acid molecules such as DNA and RNA, but also metal fine particles in which only one probe molecule for detecting biomolecules such as proteins and sugar chains is immobilized on the surface. Can be manufactured.
(1) Binding of fixed functional group to magnetic fine particle surface (2) Binding of DNA to magnetic fine particle surface (3) Formation of double-stranded DNA on magnetic fine particle surface (4) Metal to terminal functional group of probe DNA Bonding of fine particles (5) Binding of adsorption-inhibiting molecules to the surface of metal fine particles (6) Separation and collection of single probe DNA fixed metal fine particles

Each step will be described below.
(1) Binding of fixed functional group to the surface of magnetic fine particles (FIG. 3 (a))
A fixed functional group 302 for binding DNA having a base sequence complementary to the probe DNA is bound to the surface of the minute magnetic microparticle 301. The magnetic fine particle 301 has a core of a polymer blend containing a magnetic material having strong magnetization at the center, and has a structure in which a polymer such as polystyrene or polypropylene surrounds the core. Examples of the immobilizing functional group 302 for immobilizing DNA complementary to the probe DNA include a carboxyl group, streptavidin, an epoxy group, and an amino group. Binds to the surface of the magnetic particle so that chemical reactions such as hybridization that occur on the DNA bound to this fixed functional group are not hindered by steric interactions from other DNA adjacent on the surface of the magnetic particle. The density of the fixed functional group 302 to be adjusted is appropriately adjusted, and a sufficient distance is secured between the fixed functional groups.

(2) DNA binding to the surface of magnetic fine particles (FIG. 3 (b))
A DNA 303 having a base sequence complementary to the probe DNA is bound to the fixed functional group 302 on the surface of the magnetic fine particle. In a reaction vessel, a solution containing magnetic fine particles 301 and a solution containing DNA 303 are mixed and reacted. One end of the DNA 303 is modified with a terminal functional group 304 that can bind to the fixed functional group 302 on the surface of the magnetic fine particle. For example, when the fixed functional group 302 on the surface of the magnetic fine particle is a carboxyl group, the terminal functional group 304 of the DNA 303 complementary to the probe DNA is preferably an amino group. At this time, DNA303 binds to the surface of the magnetic fine particle by an amide bond. When the immobilized functional group 302 on the surface of the magnetic fine particle is streptavidin, biotin is suitable for the terminal functional group 304 of the DNA 303 complementary to the probe DNA. At this time, the DNA 303 is bound to the surface of the magnetic fine particle by an avidin-biotin bond. After the reaction, a jig with a magnet is brought close to the reaction vessel, magnetic particles are gathered near the wall of the reaction vessel, and excess DNA303 that has not bound to the fixed functional groups on the surface of the magnetic particles is washed with an appropriate buffer. Remove.

(3) Formation of double-stranded DNA on the surface of magnetic fine particles (FIG. 3 (c))
DNA303 bound to the surface of magnetic microparticles and probe DNA305 are hybridized to form double-stranded DNA. A terminal functional group 306 for binding metal fine particles is introduced at one end of the probe DNA. Examples of the terminal functional group 306 of the probe DNA include a thiol group and biotin. After the reaction, a jig with a magnet is brought close to the reaction vessel, magnetic particles are gathered near the wall of the reaction vessel, and excess probe DNA305 that did not form double-stranded DNA on the surface of the magnetic particles is placed in an appropriate buffer. Remove by washing.

(4) Binding of metal fine particles to the terminal functional group of probe DNA (FIG. 3 (d))
A single metal particle 307 is bonded to the terminal functional group 306 of the probe DNA 305 hybridized with the DNA 303 bonded to the magnetic particle. For example, when the terminal functional group of the probe DNA is a thiol group, gold nanoparticles are suitable as the metal fine particles. In addition, when the terminal functional group of the probe DNA is biotin, gold nanoparticles having streptavidin bonded to the surface as metal fine particles are preferable. After the reaction, a jig with a magnet is brought close to the reaction vessel, magnetic particles are collected on the wall of the reaction vessel, and the metal particles 307 that have not bound to the terminal functional group 306 of the probe DNA are washed away with an appropriate buffer. To do. Through the above process, single probe DNA-immobilized metal fine particles in which a single probe DNA 305 is immobilized on the surface of the metal fine particles 307 are manufactured.

  As the material of the metal fine particles 307, noble metals such as gold, silver, platinum, palladium, rhodium, iridium, ruthenium, osmium, or alloys thereof can be used. Alternatively, the surface of fine particles made of these noble metals may be used in which other noble metals are coated, for example, metal fine particles in which the surface of silver fine particles is coated with gold. The size of the metal fine particles is desirably 10 nm or more from the viewpoint of stability of fixation to the substrate surface. On the other hand, from the viewpoint of fluorescence enhancement, the size of the metal fine particles is desirably 10 nm or more and 1 m or less so that a fluorescence enhancement effect can be obtained.

  Although FIG. 3 shows an example of the probe molecule 305 having three functional groups 306 that bind to metal fine particles, a probe molecule having a plurality of functional groups that bind to metal fine particles may be used. For example, FIG. 4A shows a probe molecule 403 having a branched molecular structure and having a functional group 402 capable of binding to the metal fine particle 401 at the end of each branched chain. FIG. 4B shows a probe molecule 406 having a branched molecular structure and having a plurality of functional groups 405 capable of binding to metal fine particles 404 in the middle and at the end of each branched chain. Since the probe molecules shown in FIGS. 4A and 4B have a plurality of functional groups that can bind to the metal fine particles, the probe molecules can be more strongly bonded to the surface of the metal fine particles.

  Here, after the formation of the double-stranded DNA on the surface of the magnetic fine particles shown in step (3), the metal fine particles were bound to the terminal functional groups of the probe DNA shown in step (4). ) May be followed by step (3). That is, the probe DNA 305 and the metal fine particles 307 are mixed and reacted in a reaction container different from the reaction container containing the solution containing the magnetic fine particles 301. At this time, the ratio of the number of moles of probe DNA to the number of moles of metal fine particles is set to 1 or less, and the reaction is performed in a dilute state of probe DNA, thereby preparing a solution containing single probe DNA-immobilized metal fine particles. This solution is mixed with a solution containing magnetic fine particles 301 having DNA 303 complementary to probe DNA bonded to the surface. At this time, the DNA 303 bound to the surface of the magnetic fine particle and the probe DNA of the single probe DNA-fixed metal fine particle form a double strand. Accordingly, single probe DNA-immobilized metal fine particles can be bound to the surface of the magnetic fine particles and recovered.

(5) Binding of adsorption-inhibiting molecules to the surface of metal fine particles (FIG. 3 (e))
In the single probe DNA-immobilized metal fine particles produced in the step (4), biomolecules and the like may be adsorbed non-specifically on the surface portion of the metal fine particles to which probe molecules are not immobilized. Therefore, the adsorption-inhibiting molecule 308 is bound to the portion of the metal fine particle surface where the single probe molecule is not bound. When gold nanoparticles are used as metal fine particles, 1-mercaptohexanol, 2-mercaptoethanol, bis- (p- (sulfonatophenyl) phenylphosphine), etc. are used as adsorption-inhibiting molecules. After the reaction, a jig having a magnet is brought close to the reaction vessel, magnetic fine particles are gathered on the wall of the reaction vessel, and the adsorption-inhibiting molecules 308 that are not bound to the surface of the metal fine particles are placed in an appropriate buffer. In this case, after the binding of the metal fine particle to the terminal functional group of the probe DNA shown in step (4), the adsorption inhibiting molecule was bonded to the surface of the metal fine particle shown in step (5). , Step (4) may be performed after step (5), that is, metal fine particles having adsorption-inhibiting molecules bonded to the surface in advance are bonded to the terminal functional group of probe DNA. Good.

(6) Separation and recovery of single probe DNA-immobilized metal microparticles (FIG. 3 (f))
DNA annealing such as warming or alkali treatment is performed to dissociate the double-stranded DNA formed on the surface of the magnetic microparticles to form single-stranded DNA. At this time, a mixture of single probe DNA-immobilized metal fine particles 309 and magnetic fine particles is obtained. A jig with a magnet is brought close to the reaction vessel, magnetic particles are collected on the wall of the reaction vessel, and single probe DNA-immobilized metal particles contained in the supernatant are separated and collected.

  Next, a method for evaluating the number of immobilized DNAs of the single probe DNA-immobilized metal fine particles obtained in steps (1) to (6) will be described with reference to FIG. Methods for evaluating the number of DNA immobilized on the surface of metal fine particles include a method using gel electrophoresis and a method using a light absorption spectrum. Here, using the method disclosed in Non-Patent Document 1, the number of immobilized DNA of single probe DNA-immobilized metal fine particles is evaluated by gel electrophoresis.

(7) Evaluation of number of immobilized DNA of single-probe DNA-immobilized metal fine particles Electrophoresis gel produced with agarose, acrylamide, etc., containing a solution containing single-probe DNA-immobilized metal fine particles prepared in steps (1) to (6) Inject into 501 and perform gel electrophoresis. As a comparative sample, a solution is prepared by mixing and reacting a metal microparticle and a probe DNA having a functional group capable of binding to the surface of the metal microparticle. At this time, four types of solutions prepared by mixing and reacting so that the molar ratio of the metal fine particles to the probe DNA are 1: 0, 1: 1, 1: 2, and 1: 3, respectively. These samples for comparison include fine metal particles with probe DNA immobilized on the surface.

  The solution containing the single probe DNA-immobilized metal fine particles is placed in the well 502 of the gel 501 for electrophoresis, and the molar ratio of the metal fine particles to the probe DNA is 1: 0, 1: 1, 1: 2 and 1: 3, respectively. The mixed and reacted solutions are injected into the wells 503, 504, 505, and 506, respectively, and gel electrophoresis is started. Since the metal microparticles and probe DNA have a negative charge on the surface, when electrophoresis starts, the metal microparticles contained in each sample migrate in the electrophoresis gel from the negative electrode side 507 to the positive electrode side 508 in the direction of migration 509. To do. In addition, in the probe-immobilized metal fine particles, the effective volume during gel electrophoresis increases as the number of immobilized DNA increases, so the migration distance from the well at the migration start position becomes shorter.

  Therefore, when the comparative sample is migrated, the metal fine particles 510 on which the probe DNA is not fixed on the surface, the metal fine particles 511 on which one probe DNA is fixed on the surface, and the metal fine particles on which two probe DNAs are fixed on the surface. Electrophoretic bands corresponding to 512 and metal fine particles 513 having three probe DNAs immobilized on the surface are observed. Here, in the solution containing single-probe DNA-immobilized metal microparticles produced by steps (1) to (6), only the electrophoresis band 514 corresponding to the metal microparticles with one probe DNA immobilized on the surface is observed. The From this observation result, it can be confirmed that the single probe DNA-immobilized metal fine particles are produced with high purity by the steps (1) to (6).

  In the above description, gel electrophoresis of metal fine particles having probe DNA immobilized on the surface was performed. However, when the base length of the probe DNA is short, the increase in the effective volume of the probe-immobilized metal particles during gel electrophoresis is small even if the number of probe DNAs immobilized on the surface of the metal particles increases. Therefore, the change in the migration distance corresponding to the change in the number of immobilized probe DNAs becomes small, and it becomes difficult to separate the probe-immobilized metal fine particles. In such a case, gel electrophoresis may be performed after reacting the probe DNA-immobilized metal fine particles with a complementary molecule having a large molecular weight that can be complementarily bound to the probe DNA. As the complementary molecule, a long-chain DNA having a base sequence complementary to the probe DNA can be used. When probe-immobilized metal fine particles are reacted with complementary molecules having a large molecular weight, the complementary molecules bind to each probe DNA on the surface of the metal fine particles. That is, one, two, and three complementary molecules bind to metal fine particles having one, two, or three probe DNAs immobilized on the surface, respectively. As the number of complementary molecules bound increases, the effective volume of the probe DNA-immobilized metal particles during gel electrophoresis increases greatly, and the migration distance decreases greatly. Therefore, the migration distance of the probe DNA-immobilized metal fine particles varies greatly depending on the number of complementary molecules bound, that is, the number of immobilized probe DNA, and can be separated in the electrophoresis gel.

Next, an example of the manufacturing process of the single probe molecule element in which the single probe molecule-fixed metal fine particles of the present invention are fixed on the surface of the carrier substrate will be described with reference to FIG. Here, we will explain using DNA as a probe molecule. However, this manufacturing process is not only a nucleic acid molecule such as DNA and RNA, but also a metal fine particle in which only one probe molecule for detecting biomolecules such as proteins and sugar chains is immobilized on the surface. It is possible to manufacture a single probe molecular device in which is immobilized on the surface of a carrier substrate. The manufacturing process of a single probe molecular element in which single probe DNA-immobilized metal fine particles are fixed on the surface of a carrier substrate includes the following three steps.
(8) Formation of linker molecular layer on carrier substrate surface (9) Immobilization of single probe DNA-immobilized metal fine particles on carrier substrate surface (10) Formation of adsorption-inhibiting molecular layer on carrier substrate surface

Each step will be described below.
(8) Formation of a linker molecular layer on the surface of the carrier substrate (FIGS. 6A and 6B)
The washed carrier substrate 601 is immersed in a solution of the linker molecule A, and the linker molecule A is reacted with the surface of the carrier substrate. A glass substrate or quartz substrate is used as the substrate. As the linker molecule A, a molecule having both a functional group that binds to the substrate surface and a functional group that binds to the metal fine particles is used. For example, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-mercaptopropyltrimethoxysilane, etc. are used. As the solvent, methanol, ethanol, toluene, water or the like is used. The linker molecule A binds to the silanol group on the substrate surface, and functional groups such as amino groups and thiol groups are exposed on the opposite side of the substrate, and binds to the single probe DNA-immobilized metal particles in the next step.

(9) Immobilization of single probe DNA-immobilized metal microparticles on the surface of the carrier substrate (FIG. 6 (c))
Single probe DNA-immobilized metal microparticles 602 are immobilized on the surface of the carrier substrate by the interaction between the functional groups exposed on the surface of the carrier substrate and the metal microparticles. The functional group exposed on the surface of the carrier substrate is replaced with the adsorption-inhibiting molecule immobilized on the surface of the metal fine particle of the single probe DNA-immobilized metal fine particle, and is bonded to the surface of the metal fine particle or forms a chemical bond with the adsorption-inhibiting molecule. By this, the single probe DNA-immobilized metal fine particles are fixed on the surface of the carrier substrate. Specifically, a solution containing single probe DNA-immobilized metal fine particles is dropped on the surface of the carrier substrate and reacted at 20 to 80 ° C. for 0.5 to 50 hours. The density of the single probe DNA-immobilized metal fine particles immobilized on the surface of the carrier substrate is adjusted by adjusting the concentration of the solution containing the single probe DNA-immobilized metal fine particles dropped on the surface of the carrier substrate.

(10) Formation of adsorption-inhibiting molecular layer on the surface of the carrier substrate (FIG. 6 (d))
Of the surface of the carrier substrate, biomolecules may adsorb nonspecifically to the surface where the single probe DNA-immobilized metal microparticles are not immobilized. Biomolecules adsorbed non-specifically on the surface of the carrier substrate become detection noise and must be suppressed. Therefore, the adsorption-inhibiting molecule B is immobilized in the region where the single probe DNA-immobilized metal fine particles are not immobilized on the surface of the carrier substrate. As the adsorption-inhibiting molecule, polyethylene glycol having a terminal NHS (N-hydrosuccinimidyl) ester (NHS-PEG) or the like can be used.

  Next, an example will be described in which a single probe molecular element in which single probe DNA-immobilized metal fine particles produced through steps (1) to (10) are immobilized on the surface of a carrier substrate is used for DNA sequencing.

(11) DNA Sequencing Evaluation First, the single probe DNA-immobilized metal microparticles 701 are immobilized on the surface of the carrier substrate 702 as shown in FIG. 7 through steps (1) to (10). Next, the single DNA molecule 703 to be sequenced is reacted 1: 1 with the probe DNA 704 immobilized on the surface of the metal fine particle on the substrate, and the molecule is immobilized on the substrate surface one by one. Specifically, DNA molecules to be sequenced are dissolved in a solution containing a salt such as sodium chloride (NaCl), and this solution is dropped onto the surface of the substrate 702 on which single probe DNA-immobilized metal fine particles are fixed. The reaction temperature is 20 ° C to 80 ° C, and the reaction time is 1 to 24 hours.

  The target of actual analysis in DNA sequencing is messenger RNA (mRNA), which is a transcript of genomic DNA. The characteristic of mRNA is that it has a sequence (PolyA tail) with an average of about 200 adenine (A) at the 3 'end. In order for a single mRNA molecule to be sequenced to react with probe DNA immobilized on the surface of a metal particle, the probe DNA must have a reaction site with a single mRNA molecule. Therefore, when the sequencing target is a single mRNA molecule, a probe DNA having a PolyT sequence which is a base sequence complementary to the PolyA tail is used.

Next, as shown in FIG. 8, by using the probe DNA 802 fixed to the metal fine particles 801 as a primer, a nucleotide is extended to the tip of the probe DNA by a polymerase reaction. In FIG. 8, DNA is a molecule in which nucleotides having any one of four types of bases (adenine (A), thymine (T), guanine (G), and cytosine (C)) are continuously bound. To show that, DNA is expressed as a structure in which a large number of squares representing nucleotides are linearly connected. In the reaction shown in FIG. 8, a solution in which nucleotide 804 is dissolved in a solution containing a salt such as magnesium chloride (MgCl ₂ ) is brought into contact with the substrate. A fluorescent molecule 805 is bound to the extended nucleotide 804. By detecting the fluorescence from this fluorescent molecule, the type of the extended nucleotide can be identified. In the figure, 803 is a polymerase.

  FIG. 9 shows a DNA sequencing reading system. In this system, excitation laser light 901 is incident from the back surface of the carrier substrate 902 through the quartz prism 903 under total reflection conditions. Of the excitation laser light incident under the total reflection condition, the fluorescent molecule 909 bound to the nucleotide 908 extended by one base is excited by the evanescent light that oozes out to the surface on which the single DNA molecule 906 to be analyzed is fixed. The fluorescence 910 emitted from the fluorescent molecule 909 is measured from the upper surface of the substrate by a highly sensitive CCD camera 911. After the nucleotide is extended, the fluorescent molecule bound to the nucleotide is removed. For example, when a fluorescent molecule is bound to the end of a phosphate group of a nucleotide, the fluorescent molecule is removed from a single DNA molecule by cleavage of the phosphate group. Subsequently, another type of nucleotide is reacted. In the figure, 907 is a polymerase.

  For example, when a solution containing nucleotide A is first brought into contact with the substrate and A is extended by one base by the polymerase reaction, fluorescence is detected by the reading system described above. If nucleotide A does not extend by one base, no fluorescence is detected. From this measurement result, it can be seen that the metal fine particles in which fluorescence is detected have T complementary to A in the analysis target portion of the single DNA molecule to be analyzed. Further, it can be seen that the metal fine particles in which no fluorescence is detected do not have T in the analysis target portion. Next, for example, fluorescence is detected after bringing a solution containing nucleotide C into contact with the substrate, and it is determined whether or not each metal fine particle has G complementary to C in the analysis target portion of a single DNA molecule. The nucleotides T and G are subsequently reacted. By repeating these steps, the base sequence of the single DNA molecule 905 immobilized on each metal fine particle 904 is read. Here, as a fluorescent substance used for single base extension, a reagent in which a fluorescent dye is bound to the phosphate portion of a nucleotide is used. However, a reagent in which a fluorescent dye is bound to a purine or pyrimidine base of a nucleotide, or a 3′OH end of a nucleotide. You may use the reagent which the fluorescent dye couple | bonded.

  In steps (8) to (11), a method for producing a single probe molecule element in which the single probe molecule-immobilized metal fine particles produced in steps (1) to (7) are immobilized on the surface of the carrier substrate, and this The biomolecule detection method using was shown. On the other hand, probe molecules for detecting biomolecules, microparticles for capturing biomolecules, and biomolecules in solution without fixing the single probe molecule-fixed metal particles produced in steps (1) to (7) to the surface of the carrier substrate. It can also be used as fine particles for transportation. For example, a single probe molecule-fixed metal fine particle is taken into a cell, and a desired amount of probe molecule is delivered to a target site in the cell. In addition, it is possible to trace the transport route of probe molecules in the cell by tracking the trajectory of the metal fine particles in the cell.

  Next, a method for producing a single probe molecule-fixed metal fine particle of the present invention, a method for producing a single probe molecular device in which the metal fine particle is immobilized on the surface of a carrier substrate, and DNA sequencing using the single probe molecular device. An example is described in more detail in Example 1.

(1) Binding of fixed functional group to magnetic fine particle surface In this example, commercially available magnetic fine particles having a fixed functional group bonded to the surface were used. Specifically, commercially available carboxyl group-bonded magnetic fine particles were used. The average diameter of the magnetic fine particles was 1.05 μm. This magnetic fine particle has a core of polymer blend in which γFe ₂ O ₃ and Fe ₃ O ₄ which are magnetic materials with strong magnetization in the center are uniformly distributed, and this core is covered with an outer polystyrene layer .

(2) Binding of DNA to the surface of magnetic fine particles In this example, DNA having a base sequence of TTTTTTTTTTTTTTTTTTTT (PolyT20, base length 20) from the 5 ′ end was selected as the probe DNA. Therefore, the base sequence of DNA complementary to this probe DNA is AAAAAAAAAAAAAAAAAAAA (PolyA 20, base length 20) from the 5 ′ end side. Therefore, PolyT20 was bonded to the surface of the magnetic fine particles.

  Specifically, the magnetic fine particles were first washed with 25 mM MES (2- (N-morpholino) ethane sulfonic acid, 2- (N-morpholino) ethane sulfonic acid) buffer (pH 6). EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS (N-hydroxysuccinimidyl) ester Were dissolved in 25 mM MES buffer to adjust the concentration to 50 mg / ml. A solution containing magnetic fine particles washed with MES buffer was taken into the reaction vessel, and EDC and NHS were further added and stirred for 30 minutes. After completion of the reaction, a jig with a magnet was brought close to the reaction vessel, magnetic particles were collected on the wall of the reaction vessel, and the supernatant was removed. Next, PolyA20 having an amino group at the 5 ′ end was dissolved in MES buffer to adjust the concentration to 1 nM to 1 μM, and this was mixed and stirred with the solution containing the magnetic fine particles, and reacted at 25 ° C. for 30 minutes. . By this reaction, PolyA20 was bonded to the surface of the magnetic fine particle through an amide bond. After completion of the reaction, a jig with a magnet was brought close to the reaction vessel, magnetic particles were collected on the wall of the reaction vessel, and the supernatant was removed. Finally, among the carboxyl groups on the surface of the magnetic fine particles, the magnetic fine particles were capped with 50 mM ethanolamine (pH 8.0) prepared with PBS (Phosphate Buffered Saline) in order to capping the unbound PolyT20. Treated at ℃ for 1 hour.

(3) Formation of double-stranded DNA on the surface of magnetic fine particles In order to prevent nonspecific adsorption of probe DNA on the surface of magnetic fine particles, 0.1% to 0.5% BSA (bovine) Serum albumin (Bovine Serum Albumin) and PBS containing 0.1% surfactant (Tween-20 or Triton X-100) were washed. Next, a solution of PolyT20 (concentration 1 nM to 1 μM) in 5 × SSC (citrate buffer, Standard Saline Citrate), 0.5% SDS (Sodium Dodecyl Sulfate), which is a DNA hybridization solution Was prepared. A thiol group was introduced at the 5 ′ end of PolyT20 in order to bind metal fine particles in the next step (4). A solution containing magnetic fine particles with PolyA20 bonded to the surface was mixed with a solution of PolyT20, and hybridization was performed in a constant temperature oven at 42 ° C. for 20 hours. As a result, double-stranded DNA consisting of PolyA20 and PolyT20 was formed on the surface of the magnetic fine particles. After completion of hybridization, a jig with a magnet is brought close to the reaction vessel, magnetic particles gather on the reaction vessel wall surface, and the supernatant is removed to remove excess PolyT20 that has not hybridized with PolyA20 bound to the surface of the magnetic particles. Was removed. Further, the magnetic fine particles were washed with 5 × SSC, 0.5% SDS.

(4) Binding of metal fine particles to terminal functional groups of probe DNA Gold nanoparticles as metal fine particles were immobilized on the thiol group at the 5 ′ end of PolyT20 forming double-stranded DNA on the surface of magnetic fine particles. In this example, a gold nanoparticle citric acid solution (concentration 0.007% Weight / Volume) having a particle size of 30 nm was used. Gold nanoparticles prepared at a concentration of 1 nM to 1 μM in 5 × SSC, 0.5% SDS solution were mixed with magnetic fine particles and reacted at 25 ° C. for 16 to 24 hours. As a result, gold nanoparticles were immobilized via a thiol group on the 5 'end of PolyT20 forming double-stranded DNA on the surface of magnetic particles. After completion of the reaction, the excess gold nanoparticles that did not bind to the terminal thiol group of PolyT20 were obtained by bringing a jig with a magnet close to the reaction vessel, collecting magnetic fine particles on the reaction vessel wall, and removing the supernatant. Was removed.

(5) Binding of adsorption-inhibiting molecules to the surface of metal fine particles In the single-probe DNA-immobilized gold nanoparticles, the surface of the gold nanoparticles is not adsorbed to the portion where the probe DNA, PolyT, is immobilized. (B- (p-sulfonatophenyl) phenylphosphine) was bonded. Specifically, the solution containing magnetic fine particles prepared in steps (1) to (4) was bis- (p Add an aqueous solution of-(sulfonatophenyl) phenylphosphine and adjust the final concentration of bis- (p- (sulfonatophenyl) phenylphosphine in the reaction solution to 1 mg / ml, then react at 25 ° C for 16-24 hours did.

(6) Separation and recovery of single probe DNA-immobilized metal fine particles The solution containing the magnetic fine particles prepared in steps (1) to (5) was heated to 70 ° C. At this time, the double-stranded DNA formed on the surface of the magnetic fine particles was dissociated in step (4), and PolyA20 immobilized on the surface of the magnetic fine particles and PolyT20 immobilized on the surface of the gold nanoparticles became single-stranded DNAs. Therefore, single PolyT20-fixed gold nanoparticles and magnetic fine particles with PolyA20 fixed on the surface were released in the reaction solution. A single PolyT20-fixed gold nanoparticle was recovered by bringing a jig containing a magnet close to the reaction vessel, collecting magnetic particles on the wall of the reaction vessel, and collecting the supernatant.

(7) Evaluation of the number of DNA immobilized on single probe DNA-immobilized metal fine particles In step (6), gold nanoparticles with a particle size of 30 nm with a single PolyT20 molecule immobilized on the surface were collected. In this gold nanoparticle, since the volume occupied by PolyT20 is smaller than the volume occupied by gold nanoparticles, it was difficult to separate the gold nanoparticles by gel electrophoresis according to the number of PolyT20 immobilized on the surface. Therefore, gel electrophoresis was performed after hybridizing DNA with a longer base length to PolyT20 immobilized on the surface of gold nanoparticles. In this Example, 70mer DNA having a PolyA20 sequence at the 3 ′ end was hybridized to PolyT20 immobilized on the gold nanoparticle surface. The nucleotide sequence of 70mer DNA is from the 3 'end to the 5' end.
AAAAAAAAAAAAAAAAAAAAAGTCGAGCGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGCGGCGGACG (70mer)
It is. Specifically, single PolyT20-fixed gold nanoparticles and 70mer DNA were mixed in 5 × SSC, 0.5% SDS, and hybridized in a constant temperature oven at 42 ° C. for 20 hours.

  A solution containing single PolyT20-fixed gold nanoparticles hybridized with the 70mer DNA was injected into a well of 3% agarose gel. As a sample for comparison, a solution was prepared by reacting gold nanoparticles and PolyT20 having a 5 ′ end modified with a thiol group at molar ratios of 1: 0, 1: 1, 1: 2, and 1: 3. These comparative samples were also hybridized with the 70mer DNA and then injected into the agarose gel wells. A constant voltage (1 to 10 V / cm) was applied to the agarose gel, and gel electrophoresis of PolyT20-fixed gold nanoparticles hybridized with 70mer DNA was performed. Since double-stranded DNA consisting of gold nanoparticles and PolyT20 and 70mer DNA formed on the surface of the gold nanoparticles has a negative charge, the PolyT20-fixed gold nanoparticles contained in each sample when the electrophoresis is started are on the negative electrode side in the gel To the positive electrode side. PolyT20-immobilized gold nanoparticles form more double-stranded DNA with 70mer DNA as the number of PolyT20 immobilized on the surface increases. This increased the effective volume of PolyT20-fixed gold nanoparticles during gel electrophoresis and shortened the migration distance from the migration start position. Therefore, the difference in migration distance due to the difference in the number of PolyT20 immobilized on the gold nanoparticle surface became larger. In the electrophoresis of single PolyT20-fixed gold nanoparticles produced by steps (1) to (6), one electrophoresis band corresponding to the gold nanoparticles with one PolyT20 fixed on the surface is observed. No band was observed. Therefore, it was confirmed that single PolyT20 fixed gold nanoparticles were produced with high purity by the method disclosed in this example.

(8) Formation of linker molecular layer on quartz substrate surface The washed quartz substrate was immersed in a methanol solution of 3-aminopropyltrimethoxysilane as a silane coupling agent for 5 minutes. Subsequently, the substrate was washed with methanol and then annealed at 80 ° C. for 2 hours. This process introduced amino groups on the quartz substrate surface.

(9) Immobilization of single probe DNA-immobilized metal nanoparticles on the quartz substrate surface A solution of single PolyT20-immobilized gold nanoparticles was dropped on the quartz substrate surface into which amino groups had been introduced, and reacted at 25 ° C for 16 hours. The density of the single PolyT20 fixed gold nanoparticles fixed on the quartz substrate surface was adjusted by adjusting the concentration of the single PolyT20 fixed gold nanoparticle solution dropped on the substrate to 0.01-100 nM.

(10) Formation of adsorption-inhibiting molecular layer on substrate surface Polyethylene glycol (NHS-PEG) having NHS ester at the end as an adsorption-inhibiting molecule is applied to the region where single PolyT20-fixed gold nanoparticles are not immobilized on the carrier substrate. Combined. Specifically, the support substrate was immersed in NHS-PEG solution prepared to 4 mM with triethanolamine (pH 8.0) and reacted at 25 ° C. for 1 hour. After completion of the reaction, the carrier substrate was thoroughly washed with pure water.

(11) DNA sequencing (FIGS. 10, 11, and 12)
DNA sequencing was performed using the substrate manufactured according to steps (1) to (10). A single DNA molecule 1004 to be sequenced was hybridized to a single PolyT20 molecule 1003 immobilized on the surface of a gold nanoparticle 1002 immobilized on a quartz substrate 1001. In this example, DNA having A sequence continuously from the 3 ′ end was hybridized as a single DNA molecule to be sequenced. The base sequence of this single DNA molecule 1004 is shown from the 3 ′ end to the 5 ′ end.
AAAAAAAAAAAAAAAAAAAAAGTCGAGCGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGCGGCGGACG (70mer)

Next, the single DNA molecule 1004 was sequenced using a single PolyT20 molecule 1003 as a probe DNA as a primer. The substrate on which the single PolyT20-fixed gold nanoparticles were fixed and the four nucleotides (A, T, G, C) having the fluorescent molecule Cy3 at the phosphate group end of the nucleotide triphosphate were reacted by the polymerase reaction. . The reaction solution was a 1 μM nucleotide solution prepared with 10 mM Tris-HCl (Tris-HCl), 5 mM magnesium chloride (MgCl ₂ ) solution. A nucleotide having a base complementary to the base sequence of the single DNA molecule 1004 is bound to a single PolyT20 molecule by the polymerase reaction. In this example, when a solution of nucleotide C (indicated by 1005 in FIG. 10) is reacted with quartz substrate 1001, nucleotide C binds to a single PolyT20 molecule. This is because nucleotide C binds to G adjacent to the PolyA sequence in a single DNA molecule to be sequenced.

  Using the fluorescence detection system shown in FIG. 9, a fluorescence image during the polymerase reaction is acquired. The fluorescence intensity was measured from the top surface of the quartz substrate with a high sensitivity CCD camera. By irradiating the excitation light under the total reflection condition, the fluorescence from the gold nanoparticle 1002 to which the nucleotide C to which the fluorescent molecule Cy3 (indicated by 1006 in FIG. 10) is bound is bound is detected. After the nucleotide binds to a single PolyT20 molecule, the phosphate group of this nucleotide is cleaved. As a result, the fluorescent molecule Cy3 bound to the end of the phosphate group is removed from the single PolyT20 molecule. Next, when a solution of nucleotide A is reacted with the substrate, nucleotide A binds to T adjacent to nucleotide G of single DNA molecule 1004 to be sequenced. At this time, fluorescence is detected. By repeating these procedures, it was possible to read the base sequence of a single DNA molecule in each gold nanoparticle.

  For comparison with a quartz substrate with single PolyT20 fixed gold nanoparticles fixed on the surface, we fabricated a quartz substrate with PolyT20 molecules directly fixed on the surface. FIG. 11 (a) shows a conceptual diagram of a quartz substrate manufactured by the present embodiment, with a single PolyT20-fixed gold nanoparticle fixed on the surface, and FIG. 11 (1) shows a conceptual diagram of a quartz substrate with PolyT20 molecules directly fixed on the surface. Shown in b). In order to perform sequencing of single DNA molecules using these substrates, an extension reaction of one base contained in a single PolyT20 molecule was performed. A histogram of the fluorescence intensity of a single PolyT20 molecule obtained at this time is shown in FIG.

A quartz substrate with single PolyT20-fixed gold nanoparticles fixed on the surface (with gold nanoparticles, corresponding to FIG. 11 (a)) and a quartz substrate with PolyT20 molecules directly fixed on the surface (without gold nanoparticles, FIG. 11 ( For each of (a) and (b), the intensity of the fluorescent bright spot included in the area of about 3,000 μm ² was quantified. As a result, the total number of fluorescent bright spots was about 1,400 on the substrate without gold nanoparticles, while about 3,000 fluorescent spots were detected on the substrate with gold nanoparticles. Compared to the substrate without gold nanoparticles, the average value of fluorescence intensity was about 10 times with gold nanoparticles and the standard deviation of fluorescence intensity was halved. PolyT20 molecules directly fixed to the substrate without gold nanoparticles were fixed randomly, and the distance between adjacent PolyT20 molecules was not constant, resulting in a large variation in fluorescence intensity. On the other hand, a single PolyT20 molecule immobilized on the surface of a gold nanoparticle is immobilized on a quartz substrate surface, one molecule at a time and at a sufficient distance, and is placed under more homogeneous reaction conditions. The variation in fluorescence intensity was reduced. Furthermore, it was confirmed that the fluorescence intensity increased due to the fluorescence enhancement effect by the gold nanoparticles. Therefore, it was shown that DNA sequencing can be carried out with high accuracy and high sensitivity on the substrate having single probe DNA-immobilized gold nanoparticles immobilized on the surface in this example.

  Next, in the single probe molecular device disclosed in the present invention, the chemical structure of the probe molecule fixed to the surface of the metal fine particle through two or more chemical bonds and the single probe molecule having the probe molecule fixed to the surface An example of the manufacturing process of the fixed metal fine particles is shown in Example 2. Here, we will explain using DNA as the probe molecule, but this manufacturing process is not only a nucleic acid molecule such as DNA and RNA, but also a single probe molecule for detecting biomolecules such as proteins and sugar chains. It is possible to produce probe molecule-fixed metal fine particles.

  FIG. 13 shows the chemical structure of a probe DNA molecule having an aminodextran structure and a production method thereof. Aminodextran 1301 is a polycondensation polymer of D-glucose having an amino group 1302. In addition, aminodextran 1301 has only one aldehyde group 1303 at the reducing end. It is possible to synthesize aminodextran having various molecular weights with different degrees of polymerization of D-glucose. In this example, a process for producing a probe DNA molecule having an aminodextran structure having an average molecular weight of 70,000 will be described.

(1) Introduction of disulfide group into aminodextran Aminodextran 1301 (average molecular weight 70,000) was dissolved in 1 × PBS (phosphate buffered phosphate, pH 7.0 to 9.0). In addition, SPDP molecule 1304 (3- (2-Pyridyldithio) propionic acid N-hydrosuccinimide ester) was dissolved in dimethylsulfoxide to prepare a concentration of 10 mM to 500 mM. The SPDP solution was mixed with the aminodextran solution and reacted at room temperature with stirring for 0.5 to 20 hours. By this reaction, aminodextran 1305 having a disulfide group 1306 introduced therein was obtained. After completion of the reaction, unreacted SPDP molecules 1304 contained in the reaction solution were removed by dialysis or gel filtration of the reaction solution.

(2) Binding of aminodextran and single probe DNA molecule The probe DNA molecule 1308 whose end was modified with an amino group 1307 was dissolved in 1 × PBS (phosphate buffer, Phosphate Buffered Saline, pH 7.0 to 9.0). A reaction solution was prepared by mixing the solution of aminodextran 1305 into which the disulfide group was introduced and the solution of the probe DNA molecule 1308. The mixing molar ratio of aminodextran 1305 having a disulfide group introduced and probe DNA molecule 1308 having a terminal modified with an amino group was in the range of 1: 1 to 1: 100. Further, a solution of NaCNBH ₃ (Soduim cyanoborohydride) dissolved in 1M NaOH (sodium hydroxide) was added to the reaction solution, and reacted for 2 to 20 hours with stirring at room temperature. By this reaction, the aldehyde group at the reducing end of aminodextran 1305 into which disulfide group was introduced and the terminal amino group 1307 of probe DNA molecule 1308 formed an amide bond, and aminodextran and probe DNA molecule were combined at 1: 1. A dextran probe DNA molecule 1309 was obtained. After completion of the reaction, unreacted probe DNA molecules 1308 contained in the reaction solution were removed by dialysis or gel filtration of the reaction solution.

  Next, FIG. 14 shows a schematic diagram of single probe DNA molecule-fixed metal fine particles in which dextran-type probe DNA molecules are fixed on the surface. In this example, according to the method shown in steps (1) to (4) of Example 1, dextran-type probe DNA molecule 1401 (1401 is a simplified version of 1309) was converted to gold nanoparticle having a particle diameter of 10 nm. A single probe molecule-fixed gold nanoparticle 1403 was obtained by binding to the surface of the particle 1402. In this example, a large number of disulfide bonds of the dextran-type probe DNA molecule 1401 are cleaved, and a large number of sulfur atoms 1404 are bonded to the surface of the gold nanoparticle 1402.

Like the dextran-type probe DNA molecule 1401 shown in this example, a probe molecule immobilized on the surface of a metal microparticle through a large number of chemical bonds is immobilized on the surface of the metal microparticle through a single chemical bond. Compared to probe molecules, there are the following advantages.
(A) By optimizing the molecular weight of the polymer portion of the probe molecule (aminodextran in this example) corresponding to the particle size of the metal fine particle to be used, a single unit in which the metal fine particle and the probe molecule are combined at 1: 1. It is possible to obtain probe molecule fixed metal fine particles with high yield.
(B) Since the probe molecule and the metal fine particles are firmly bonded via a large number of chemical bonds, the probe molecule and the metal fine particles are not easily dissociated and stable even if the temperature, ionic strength, pH, etc. of the solution change. .
(C) When the polymer portion of the probe molecule has a large number of hydrophilic functional groups (in this example, the hydroxyl group of aminodextran), the surface of the single probe molecule-fixed metal fine particle is coated with a large number of hydrophilic functional groups. Therefore, nonspecific adsorption of biomolecules is suppressed.

  The element of the present invention is applied as a biomolecule detection element for high-precision and high-sensitivity detection at the single molecule level in DNA microarrays for comprehensive quantitative analysis of nucleic acids and DNA sequencers for decoding nucleic acid base sequences. it can. Further, the element of the present invention can be used as a detection element for detecting biomolecules such as proteins and sugar chains and various chemical substances with high accuracy and high sensitivity using a fluorescence method.

The schematic diagram which shows a single probe molecule fixed metal microparticle. The schematic diagram which shows the biomolecule detection element which fixed the single probe molecule fixed metal fine particle to the support substrate surface. The schematic diagram which shows the method of manufacturing a single probe molecule fixed metal microparticle using magnetic microparticles. The schematic diagram which shows the single probe molecule | numerator couple | bonded with the surface of the metal microparticle through several functional group. The schematic diagram which shows the method of isolate | separating a probe molecule fixed metal microparticle by gel electrophoresis. The schematic diagram which shows the method of manufacturing a biomolecule detection element by fixing a single probe molecule fixed metal microparticle to the support substrate surface. The schematic diagram which shows the state which fixed the single DNA molecule which is a sequencing object to the biomolecule detection element which fixed the single probe DNA fixed metal microparticle to the support substrate surface. Schematic diagram showing a single probe DNA for DNA sequencing and a single DNA molecule to be sequenced. FIG. 2 is a schematic diagram showing a DNA sequencing reader. The schematic diagram which shows the single-base extension reaction of DNA sequencing. (A) The biomolecule detection element which fixed the single PolyT20 fixed gold nanoparticle on the quartz substrate surface, and (b) The schematic diagram which shows the biomolecule detection element which fixed PolyT20 molecule | numerator directly on the quartz substrate surface. Histogram of fluorescence intensity of each single PolyT20 molecule detected during a single base extension reaction of DNA sequencing. The schematic diagram which shows the chemical structure of the probe DNA molecule which has an aminodextran structure, and a manufacturing method. The schematic diagram which shows the single probe DNA molecule fixed metal microparticle which fixed the dextran type probe DNA molecule on the surface.

Explanation of symbols

101: single probe molecule, 102: metal fine particle, 103: adsorption inhibition molecule, 201: carrier substrate, 202: single probe molecule fixed metal fine particle, 301: magnetic fine particle, 302: fixed functional group, 303: complementary to probe DNA 304: terminal functional group, 305: probe DNA, 306: terminal functional group, 307: metal fine particle, 308: adsorption-inhibiting molecule, 309: single probe DNA-fixed metal fine particle, 401: metal fine particle 402: Functional group that binds to metal fine particles, 403: Single probe molecule, 404: Metal fine particle, 405: Functional group that binds to metal fine particle, 406: Single probe molecule, 501: Gel for electrophoresis, 502: Electricity Electrophoresis gel well, 503: Electrophoresis gel well, 504: Electrophoresis gel well, 505: Electrophoresis gel well 506: Well of electrophoresis gel, 507: Negative electrode side of electrophoresis gel, 508: Positive electrode side of electrophoresis gel, 509: Electrophoresis migration direction, 510: Metal fine particles on which DNA is not fixed, 511: Metal fine particles with one DNA fixed on the surface, 512: Metal fine particles with two DNA fixed on the surface, 513: Metal fine particles with three DNA fixed on the surface, 514: Single probe DNA fixed metal Electrophoretic band corresponding to fine particles, 601: carrier substrate, 602: single probe DNA fixed metal fine particles, 701: metal fine particles, 702: carrier substrate, 703: single DNA molecule to be sequenced, 704: single probe DNA, 801: metal fine particle, 802: single probe DNA, 803: polymerase, 804: nucleotide, 805: fluorescent molecule, 901: excitation laser light, 90 : Carrier substrate, 903: quartz prism, 904: fine metal particles, 905: single probe DNA, 906: single DNA molecule to be sequenced, 907: polymerase, 908: nucleotide, 909: fluorescent molecule, 910: fluorescent molecule , Fluorescence emitted from 911, high sensitivity CCD camera, 1001: carrier substrate, 1002: fine metal particles, 1003: single PolyT20 molecule, 1004: single DNA molecule to be sequenced, 1005: nucleotide C, 1006: fluorescent molecule Cy3, 1101: single PolyT20 fixed gold nanoparticle, 1102: quartz substrate, 1103: single PolyT20 molecule, 1104: quartz substrate, 1301: aminodextran, 1302: amino group, 1303: aldehyde group, 1304: SPDP molecule, 1305 : Aminodextran having a disulfide group introduced, 1306: Di Rufido group, 1307: amino group, 1308: probe DNA molecule modified with an amino group at the end, 1309: dextran probe DNA molecule, 1401: dextran probe DNA molecule, 1402: gold nanoparticle, 1403: single probe molecule Fixed gold nanoparticles, 1404: sulfur atom.

Claims (14)

A substrate,
A plurality of fine metal particles fixed to the surface of the substrate;
Each probe molecule has a branched chemical structure, and each probe molecule has a branched chemical structure, characterized in that each probe molecule has a probe molecule fixed to the surface via a chemical bond . A single probe molecular device having a plurality of functional groups capable of binding to metal fine particles in the middle and at the end of a branched chain, and being fixed to the surface of one metal fine particle.   2. The single probe molecular device according to claim 1, wherein the surface of the metal fine particle is coated with an adsorption inhibiting molecule in which a region other than the region in which the probe molecule is fixed is fixed through a chemical bond. Feature single probe molecular device.   2. The single probe molecular device according to claim 1, wherein the surface of the substrate is covered with an adsorption inhibiting molecule in which a region other than the region in which the metal fine particles are fixed is fixed through a chemical bond. Single probe molecular device.   2. The single probe molecular device according to claim 1, wherein the fine metal particles are made of a single layer of a metal belonging to a noble metal, a metal alloy belonging to a noble metal, or a metal belonging to a noble metal. Probe molecular element.   2. The single probe molecular device according to claim 1, wherein the probe molecule is a nucleic acid. Fine metal particles,
In single probe molecule fixing metal fine particles characterized by having a single probe molecules immobilized through a chemical bond to the surface of the fine metal particles, wherein the probe molecules have a branched chemical structure, each branch A single probe molecule-fixed metal fine particle having a plurality of functional groups capable of binding to metal fine particles in the middle and at the end of the chain, and being fixed to the surface of the metal fine particles via the functional group. 7. The single probe molecule-fixed metal fine particle according to claim 6 , wherein the surface of the metal fine particle is coated with an adsorption inhibiting molecule in which a region other than the region where the probe molecule is fixed is fixed through a chemical bond. A single probe molecule-fixed metal fine particle characterized by the above. 7. The single probe molecule-fixed metal fine particle according to claim 6 , wherein the metal fine particle is formed by laminating a metal belonging to a noble metal, a metal alloy belonging to a noble metal, or a metal belonging to a noble metal. Single probe molecule fixed metal fine particles. The single probe molecule-fixed metal microparticle according to claim 6 , wherein the probe molecule is a nucleic acid. Binding a complementary molecule that binds complementarily to the probe molecule to the surface of the magnetic particle;
A step of complementarily binding the probe molecule capable of binding to the metal fine particle to the complementary molecule;
Binding metal fine particles to the probe molecules bound to the complementary molecules;
Dissociating the complementary bond between the complementary molecule bound to the surface of the magnetic fine particle and the probe molecule;
Recovering fine metal particles having a single probe molecule immobilized on the surface;
Fixing the recovered metal fine particles on a carrier substrate;
A single probe for each of the plurality of metal fine particles immobilized on the substrate surface, wherein the adsorption inhibiting molecule is immobilized on a region of the surface of the carrier substrate where the metal fine particles are not immobilized. A method for producing a single probe molecular device having molecules immobilized thereon. Binding a complementary molecule that binds complementarily to the probe molecule to the surface of the magnetic particle;
A step of mixing and reacting the probe molecules and metal fine particles under the condition that the ratio of the number of moles of probe molecules to the number of moles of metal fine particles is 1 or less ;
The complementary molecule bonded to the surface of the magnetic fine particle is complementarily bonded to the probe molecule fixed to the surface of the metal fine particle, and the metal fine particle on which the single probe molecule is fixed is attached to the surface of the magnetic fine particle. A process of collecting;
Dissociating the complementary bond between the complementary molecule bound to the surface of the magnetic fine particle and the probe molecule fixed to the surface of the metal fine particle;
Separating and recovering fine metal particles having a single probe molecule immobilized on the surface from the magnetic fine particles;
Fixing the separated and collected metal fine particles on a carrier substrate;
A step of fixing adsorption-inhibiting molecules to a region of the surface of the carrier substrate where the metal fine particles are not fixed. Each of the plurality of metal fine particles fixed on the substrate surface has a single probe molecule. Of manufacturing a single probe molecular device with a fixed surface. Binding a complementary molecule that binds complementarily to the probe molecule to the surface of the magnetic particle;
A step of complementarily binding the probe molecule capable of binding to the metal fine particle to the complementary molecule;
Binding metal fine particles to the probe molecules bound to the complementary molecules;
Dissociating the complementary bond between the complementary molecule bound to the surface of the magnetic fine particle and the probe molecule;
Recovering fine metal particles having a single probe molecule immobilized on the surface;
A method for producing fine metal particles having a single probe molecule immobilized thereon, comprising: Binding a complementary molecule that binds complementarily to the probe molecule to the surface of the magnetic particle;
A step of mixing and reacting the probe molecules and metal fine particles under the condition that the ratio of the number of moles of probe molecules to the number of moles of metal fine particles is 1 or less ;
The complementary molecule bonded to the surface of the magnetic fine particle is complementarily bonded to the probe molecule fixed to the surface of the metal fine particle, and the metal fine particle on which the single probe molecule is fixed is attached to the surface of the magnetic fine particle. A process of collecting;
Dissociating the complementary bond between the complementary molecule bound to the surface of the magnetic fine particle and the probe molecule fixed to the surface of the metal fine particle;
Separating and recovering fine metal particles having a single probe molecule immobilized on the surface from the magnetic fine particles;
A method for producing fine metal particles having a single probe molecule immobilized thereon, comprising: The surfaces of the metal fine particles their respective fixed to the substrate surface a single probe molecule elements single probe molecule is fixed via a chemical bond, each probe molecule has a branched chemical structure, In the middle and at the end of each branched chain, there are a plurality of functional groups capable of binding to metal fine particles, and the probe molecules in the single probe molecular element fixed on the surface of one metal fine particle are to be sequenced. Reacting with a nucleic acid molecule;
Perform single base extension reactions of the probe component element, the step of reacting a fluorescent-labeled nucleotide into the nucleic acid molecule is a sequencing object,
Irradiating the single probe molecular element with excitation light;
And a step of detecting fluorescence generated from the fluorescence-labeled nucleotide.

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